Op0056 Deciphering the Crosstalk Between Cd74+ Synovial Fibroblasts and Immune Cells in Rheumatoid Arthritis

Background: Distinct synovial fibroblasts (SF) subtypes in the synovium of patients with rheumatoid arthritis (RA) have been previously described. While there is evidence of their immune–effector properties, their interaction with infiltrating immune cells and their role in the inflammatory process remain poorly understood. The contribution of immune-effector SF subsets may be involved in poor patient response to treatment. Objectives: To identify a SF subtype with potential pro-inflammatory properties and unravel its crosstalk with immune cells. Methods: Synovial tissue from RA patients who underwent joint replacement surgery was formalin-fixed and paraffin-embedded for immunohistochemistry (IHC; CXCL12, SPP1; n=28) or enzymatically and mechanically digested (n=3). Analysis of IHC was performed with QuPath. To determine the pro-inflammatory SF subtype, we isolated three SF subtypes (CD90-, CD90+CD74-, CD90+CD74+) using fluorescence-activated cell sorting and measured IL-6, Pro-Collagen1a1 and MMP3 secretion with ELISA. Healthy Monocytes were differentiated into monocyte-derived Macrophages (MDM) using M-CSF. Each SF subtype was directly co-cultured with MDMs. The expression of the activation marker CD80 in MDMs was assessed by flow cytometry. To determine which component contributes to the pro-inflammatory SF phenotype, we directly co-cultured RA SFs (n=3; > passage 3) with RA MDMs, RA T-cells, or stimulated with RA synovial fluid. CD74 expression was assessed after 48 hours by flow cytometry and qPCR. To recapitulate the synovial microenvironment, we incorporated SFs that acquired CD74 expression into a 3D synovial organoid model with HUVECs and MDMs (n=3). After 14 days, organoids were disaggregated, and MDM activation was assessed by flow cytometry based on the classical and alternative activation markers: CD80, CD40, CD38, CD206, CD163. Results: In the sublining layer, CXCL12+CD74+ SFs were found near the inflammatory infiltrates and the pro-inflammatory SPP1+ Macrophages (MCs) (Figure 1A). CD74+ SFs were higher in patients with a lymphoid pathotype (p-value 0.034) and correlated with the presence of CD3+ cells (R squared 0.147, p-value 0.04). After isolation, CD74+ SFs demonstrated increased expression of IL-6 after 48 hours (Fold Change (FC): 4.22 vs CD90- SF) but decreased levels of MMP3 and Pro-Collagen 1a1 compared to CD90- SF and CD90+CD74- SF, respectively (Figure 1B). Healthy MDMs co-cultured with CD74+ SFs demonstrated an increased expression of CD80 (FC: 12.86 vs CD90- SF). These data suggest that the CD74+ SF subtype has pro-inflammatory properties. This effect was lost after 6 days in culture and CD74+ SFs lost their CD74 expression. While co-culture with RA MDMs and stimulation with RA synovial fluid did not induce CD74 expression, co-culture with RA T-cells resulted in the acquisition of CD74 expression (FACS; p-value: 0.001). Upon incorporation into the 3D synovial organoid model, cultured SFs with acquired CD74 expression maintained their distinctive phenotype and induced an activated phenotype in healthy MDMs as shown by increased expression of CD80, CD40, CD38 (FC: 0.62, 1.93, 3.37 vs organoids with CD74- SFs, respectively). MDMs had decreased expression of CD206 (FC: -0.2 vs organoids with CD74- SFs) akin to MCs in treatment-naive/resistant RA patients1. Conclusion: Our findings suggest that the CD74+ SF subtype plays a role in shaping inflammatory niches, where activated MCs are present. This phenotype is associated with promoting synovial inflammation, particularly after interacting with infiltrating T-cells. This interaction imparts pro-inflammatory properties to the CD74+ SFs and enables them to activate MDMs. REFERENCES: 1 Alivernini S. et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nat Med. (2020). Acknowledgements: FOREUM - Foundation for Research in Rheumatology. Disclosure of Interests: Melpomeni Toitou: None declared, Eva Camarillo: None declared, Stacey Grealis: None declared, Andrea Laimbacher: None declared, Oliver Distler OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., OD has/had consultancy relationship with and/or has received research funding from and/or has served as a speaker for the following companies in the area of potential treatments for systemic sclerosis and its complications in the last three calendar years: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB. Patent issued "mir-29 for the treatment of systemic sclerosis" (US8247389, EP2331143). Co-founder of CITUS AG., Caroline Ospelt: None declared.